Serotonin (5-hydroxytryptamine, 5HT) mediates a wide variety of central and peripheral psychological and physiological effects through 14 mammalian 5HT receptor subtypes that are grouped into the 5HT1-5HT7 families (Sanders-Bush and Mayer, 2006). The 5HT2 family consists of the 5HT2A, 5HT2B, and 5HT2C membrane-bound G protein-coupled receptors (GPCRs) that signal primarily through Gαg to activate phospholipase (PL) C and formation of inositol phosphates (IP) and diacylglycerol (DAG) second messengers (Raymond et al., 2001). The human 5HT2C receptor (Saltzman et al., 1991) apparently is found exclusively in brain where it is widely expressed and putatively involved in several (patho)-physiological and psychological processes, including, ingestive behavior (Tecott et al., 1995), cocaine addiction (Fletcher et al., 2002; Rocha et al., 2002; Muller and Huston, 2006), sleep homeostasis (Frank et al., 2002), anxiety (Kennett et al., 1994; Sard et al., 2005; Heisler et al., 2007), depression (Tohda et al., 1989; Palvimaki et al., 1996), epilepsy (Heisler et al., 1998), Alzheimer's disease (Arjona et al., 2002; Stein et al., 2004), motor function (Heisler and Tecott, 2000; Segman et al., 2000), psychosis (Marquis et al., 2007; Siuciak et al., 2007) and response to antipsychotic drugs (Veenstra-VanderWeele et al., 2000; Reynolds et al., 2005). Thus, the importance of the 5HT2C receptor as a pharmacotherapeutic target has been apparent for about 10 years, however, no 5HT2C-specific drugs have been developed.
One challenge regarding drug discovery targeting the 5HT2C receptor is that this GPCR shares a transmembrane domain (TMD) sequence identity of about 80% with the 5HT2A receptor and about 70% with the 5HT2B receptor (Julius et al., 1988; 1990). The highly conserved TMDs and similar second messenger coupling has made development of agonist ligands selective for the 5HT2C receptor especially difficult. Nevertheless, there is compelling evidence that activation of 5HT2C receptors reduces food intake and leads to anti-obesity effects. For example, 5-HT2C knockout mice demonstrate increased feeding and obesity, and, they are resistant to the anorectic effects of d-fenfluramine (Tecott et al., 1995; Vickers et al., 1999; 2001; Heisler et al., 2002). Fenfluramine now is banned, because, although people using the drug showed weight loss due to activation of brain 5HT2C receptors, fenfluramine also activates 5HT2A receptors that may lead to adverse psychiatric (hallucinogenic) effects (Nichols, 2004) and 5HT2B receptors which causes valvular heart disease (Connolly et al., 1997; Fitzgerald et al., 2000; Rothman et al., 2000; Roth, 2007) and pulmonary hypertension (Pouwels et al., 1990; Launay et al., 2002)—fatalities have resulted from the 5HT2B-mediated effects.
Although an agonist ligand truly selective for 5HT2C vs. 5HT2A and/or 5HT2B receptors has not been reported until this paper, it has been possible to partially elucidate the role of brain 5HT2C receptors to attenuate cocaine use and dependence using very selective (i.e., at least 100-fold) 5HT2A and 5HT2C antagonists in rat cocaine self-administration paradigms. For example, the selective 5HT2A antagonist M100907 (Kehne et al., 1996) does not alter responding rate for cocaine self-administration but the selective 5HT2C antagonist SB242084 (Bromidge et al., 1997) increases the rate of cocaine self-administration dose-dependently (Fletcher et al., 2002). The tremendous potential of 5HT2C agonist pharmacotherapy for psychostimulant addiction now is widely recognized (Bubar and Cunningham, 2006).
The pharmacotherapeutic relevance of the 5HT2C receptor in obesity and neuropsychiatric disorders such as psychostimulant addiction has stimulated intense interest by pharmaceutical companies to develop a selective 5HT2C agonist, however, all 5HT2C agonists reported so far also activate 5HT2A and/or 5HT2B receptors (Nilsson, 2006). Nevertheless, the 5HT2 agonist lorcaserin (APD356) recently went to Phase III clinical trials for obesity treatment even though it has only a modest 15-fold selectivity for activation of 5HT2c receptors over 5HT2A receptors (Jensen, 2006; Smith et al., 2006). Results reported here, however, are based on novel compounds synthesized in our laboratories, i.e., phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (PAT) and cyclyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (CAT) compounds, are full efficacy agonist at human 5HT2C receptors, plus, it is an antagonist at 5HT2A and 5HT2B receptors.
G Protein-Coupled Receptors (GPCRs) can activate more than more type of G protein that results in multiple physiological/pharmacological effects, both pharmacotherapeutic and untoward side effects (Moniri et al., Journal of Pharmacology and Experimental Therapeutics, 311:274-281 (2004)). The phenomenon of multiple signaling pathways associated with a single GPCR can be described within the framework of the three-state model of GPCR activation, wherein, GPCRs isomerize between inactive and constitutively active states. GPCR activation causes dissociation of heterotrimeric (α,β,γ) G protein subunits—the Gα subunit can then activate transducer protein (e.g., PLC, AC) to alter second messenger concentration. It is now realized the same GPCR can couple to different Gα proteins to result in “multifunctional signaling”. A critical assumption of the GPCR multifunctional signaling theory is that a heterogeneity of active receptor conformations exists and that agonist ligands differ in their ability to induce, stabilize, or select among receptor conformations, as described in the “stimulus trafficking” hypothesis. It follows that, upon binding, agonist ligand chemical structural parameters are among the most important determinants of GPCR conformation that influences type of Gα protein and signaling pathway activated.
A survey of 105 articles on the activity of 380 antagonists on 73 biological G-protein-coupled receptor targets indicates that, in this sample dataset, 322 are inverse agonists and 58 (15%) are neutral antagonists. The predominance of inverse agonism agrees with theoretical predictions which indicate that neutral antagonists are the minority species in pharmacological space (Kenakin, Mol Pharmacol. (2004); 65:2-11).
The p90 ribosomal s6 kinases (RSKs) are a group of serine/threonine kinases that are constituents of the AGC subfamily in the human kinome (Nguyen, 2008). There are four RSK isoforms (RSK1-4) and each is a product of a separate gene. The RSK isoforms are characterized by 75%-80% sequence identity. Although the RSK isoforms are broadly distributed in human tissue, they exhibit variable tissue expression, which is an indication that they may be involved in different functions. The RSK isoforms are activated by extracellular signaling molecules that stimulate the Ras-ERK pathway. These molecules include a variety of different growth factors, cytokines, peptide hormones and neurotransmitters. It is noted that at this time, biological/pharmacological roles for each of the RSK isoforms is not established for most diseases, including, cancer.
RSKs phosphorylate a variety of proteins, including transcription factors, immediate-early gene products, translational regulators, enzymes, and structural proteins, that link RSKs as a family to many biological processes such as cell proliferation, cell differentiation, survival, and migration (Anjum and Blenis, 2008a).
RSK isoforms are overexpressed in 50% of human breast cancer tissue samples, indicating that regulation of RSK has been compromised—the link between RSK activity and tumor cell proliferation reveals RSKs as novel cancer drug targets (Gioeli et al., 1999; Smith et al., 2005).
There is compelling evidence for a link between RSKs and HIV infection. RSK2 was shown to have a reciprocal relationship with HIV-1 Tat. RSK2 is recruited and activated by HIV-1 Tat, and is itself also important to normal Tat function (Hetzer et al., 2005). Moreover, Kaposi sarcoma-associated herpesvirus interacts with RSKs and strongly stimulates their kinase activities (Kuang et al., 2009).
Structurally, the RSK isoforms are very similar (Nguyen, 2008). Each of the isoforms contains two functional catalytic domains that are separated by a large 100 amino acid linker region. The two kinase domains are distinct and contain nonidentical ATP-binding site. The NTKD is similar to p70 S6 kinase (p70 S6K1), and the CTKD is similar to the calcium/calmodulin protein kinases. Because of its molecular architecture, RSK offers two logical sites for inhibition: the ATP-binding site in the NTKD and the one in the CTKD. Site specific inhibitors for both ATP pockets have been identified.
Aberrant activation of the upstream activators of 90 kDa ribosomal S6 kinase (RSK) has been linked to many human diseases, including cancers. The RSK1 and RSK2 isoforms are amplified in breast and prostate tumours. The importance of RSK2 activation in haematopoietic transformation was recently shown in fibroblast growth factor receptor-3 (FGFR3)-expressing multiple myeloma cells. RSK3 was recently shown to function as a potential tumour suppressor in ovarian cancer. RSK4 was shown to participate in p53-dependent cell growth arrest, although aberrant expression of RSK4 has been observed in breast cancer. Mutations in the human RSK2 gene are associated with Coffin-Lowry syndrome (CLS), an X-linked disorder that is characterized by severe psychomotor retardation, digit and facial dysmorphisms, and progressive skeletal deformations. In fibroblasts from CLS patients, a drastic attenuation in induced Ser133 phosphorylation of the transcription factor CREB was detected in response to epidermal growth factor stimulation. Fibroblasts that were derived from patients with CLS have been useful in determining the function of RSK2 with respect to this human disease; however, differences found between these cells and RSK2-deficient mouse fibroblasts suggest that CLS might be a multivariable disease and mouse might not be the ideal system in which to study RSK2 function. Both the RSK2 and RSK4 genes are located on chromosome X, and recent data also implicate RSK4 in nonspecific X-linked mental retardation, but definitive evidence remains to be provided for RSK4 (Anjum and Blenis, 2008b).